**3.** *Mycobacterium pinnipedii* **infections in** *Arctocephalus australis***,** *Otaria flavescens* **and** *Mirounga leonina* **individuals**

Bacteria belonging to the Genera *Mycobacterium* are acid-fast bacilli (AFB) classified in different complexes and species according to biochemical, cultural and genetic features. The first communication of tuberculosis (TB) in captive seals dates from the early twentieth century (Blair, 1913). However, the diagnosis and study of tuberculosis and mycobacteriosis in different species of marine mammals is fairly recent. Ehlers (1965) reported a tuberculosis case in a Northern seal (*Cystophora cristata*). Subsequently, Kinne (1985) described tuberculosis cases in several marine mammal species. In 1986, the first isolates of *Mycobacterium* spp. were obtained in Australian fur seals and sea lions (*Arctocephalus pusillus doriferus* and *Neophoca cinerea* respectively) and New Zealand fur seals (*Arctocephalus forsteri*) in captive and wild conditions (Forshaw & Phelps, 1991; Woods et al., 1995). Successive isolations were made from wild pinniped species in the Southern Hemisphere (Bastida et al., 1999; Bernardelli et al., 1996; Cousins et al., 1993; Hunter et al., 1998; Romano et al., 1995; Woods et al., 1995; Zumárraga et al., 1999).

In Uruguay, the first isolation of *Mycobacterium* spp. in pinnipeds was conducted in 1987 from samples of South American sea lion *O. flavescens* specimens kept in "Villa Dolores" municipal zoo. Of the ten animals studied, one died and nine others were tuberculinpositive and were later euthanized. Seven animals showed typical histo-pathological lesions of tuberculosis, and a total of 6 strains were isolated. Initially, it was considered that the bacillus was *M. bovis* according to results from a smear, biochemical tests and culture features. The strains were inoculated to guinea pigs (0,1 mg) that developed characteristic lesions and subsequently *Mycobacterium* spp. were isolated, fulfilling Koch's postulates (Castro-Ramos et al., 1998). In 1997 *Mycobacterium* spp. was isolated from lung samples of an adult South American fur seal stranded on the coast of Montevideo. The animal was collected from the beach by a NGO and sent to quarantine in a zoo for recovery, but died four days after admission. The observed granulomatous lung lesions were typical of TB, and a *Mycobacterium* spp. strain was isolated (Castro-Ramos et al., 2001).

162 New Approaches to the Study of Marine Mammals

and B/Sichuan/379/99.

% of samples

epidemic diseases.

**Figure 6.** Antibodies to Influenza A and B virus by HAI in fur seal sera. Percent (number

<80 80 160 320 640 HAI titer

Titers ≥ 80 were considered positive. Antigens used: A/New Caledonia/20/99(H1N1),

*flavescens* **and** *Mirounga leonina* **individuals** 

Woods et al., 1995; Zumárraga et al., 1999).

positive/number tested) of samples bearing antibodies vs. HAI titer for each Influenza antigen assayed.

A/H1N1 A/H3N2 B/Beijing B/Hong Kong B/Sichuan

Monitoring the distribution of the influenza virus in wild animal species including marine mammals is important for understanding the ecology and evolution of the virus, and also to understand how the virus can mutate and re-emerge more virulent, producing devastating

**3.** *Mycobacterium pinnipedii* **infections in** *Arctocephalus australis***,** *Otaria* 

Bacteria belonging to the Genera *Mycobacterium* are acid-fast bacilli (AFB) classified in different complexes and species according to biochemical, cultural and genetic features. The first communication of tuberculosis (TB) in captive seals dates from the early twentieth century (Blair, 1913). However, the diagnosis and study of tuberculosis and mycobacteriosis in different species of marine mammals is fairly recent. Ehlers (1965) reported a tuberculosis case in a Northern seal (*Cystophora cristata*). Subsequently, Kinne (1985) described tuberculosis cases in several marine mammal species. In 1986, the first isolates of *Mycobacterium* spp. were obtained in Australian fur seals and sea lions (*Arctocephalus pusillus doriferus* and *Neophoca cinerea* respectively) and New Zealand fur seals (*Arctocephalus forsteri*) in captive and wild conditions (Forshaw & Phelps, 1991; Woods et al., 1995). Successive isolations were made from wild pinniped species in the Southern Hemisphere (Bastida et al., 1999; Bernardelli et al., 1996; Cousins et al., 1993; Hunter et al., 1998; Romano et al., 1995;

A/Panamá/2007/99(H3N2), B/Beijing/184/93-like viruses, B/Yamanashi/166/98, B/Hong Kong/330/01,

Between 2001 and 2006, pathological, microbiological and genetic studies were conducted on dead stranded animals of different species of pinnipeds found along ocean shores of Uruguay: South American fur seal (n = 129), South American sea lion (n = 24) and Southern elephant seal (n = 1). Necropsies were performed using standard methods (Dierauf, 1990). Samples from several organs with or without lesions (lung, mediastinal lymph nodes, spleen, liver) (n = 36) were stored at 4° C, frozen at -20° C or fixed in 10% formalin.

Formalin fixed samples were processed by standard histological methods: 4-5 cuts were made at 5-6 µm and stained with Hematoxylin-Eosin and Ziehl-Neelsen (ZN) (Luna, 1968). Mycobacteriological studies were performed according to the methodology described by the Pan American Zoonoses Center (Centro Panamericano de Zoonosis (OPS/OMS) (1979), Office International des Epizooties (OIE) (2000), Runyon et al., (1980) and Tacquet et al., (1967). Smears from single or pooled samples of each animal were performed and then cultured in Stonebrink and Lowenstein Jensen media. Cultures were kept for eight weeks at 37° C and periodically reviewed. Culture tests were based on microscopic features, morphology of the colony, growth temperature, time of development and cromogenicity of isolates. The identification was completed with the following biochemical tests: niacin, nitrate reduction, catalase at 22° C and 68° C, hydrolysis of Tween 80 at 5 and 10 days, reduction of potassium tellurite 0.2% at 3 days, urease and pyrazinamidase. A total of 14 strains were isolated (Table 1).

Strains isolated in 1987 (N° 01073, adult male *O. flavescens*), 1997 (N ° 01337, juvenile male *A. australis*) and 2002 (N ° 2493, juvenile female *O. flavescens*) were analyzed through amplification of 200 bp of the Internal Transcribed Spacer (ITS) region through Polymerase Chain Reaction (PCR) as in Roth et al., (2000). Sequences obtained were compared to those available at GenBank database through a maximum parsimony phylogenetic tree, and strains were grouped with sequences of *M. tuberculosis / M. pinnipedii*.

During the necropsies, granulomatous lesions were observed in only five animals: two South American sea lions (juvenile male and female) and three South American fur seals (two adult and one juvenile male) (Fig. 7), from which *M. tuberculosis / M. pinnipedii* complex

strains were isolated. Isolates were also obtained from organs without gross lesions belonging to pups and juvenile fur seals (n = 9) and from a sub-adult male elephant seal (Castro-Ramos et al., 2005, 2006) (Fig. 9).

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 165

**Figure 7.** Macroscopic view of an *Arctocephalus australis* lung. Numerous granulomas were present on the surface and deep parenchyma (white arrows) and a significant thickening of the visceral pleura.

**Figure 8.** Histological section from an *Arctocephalus australis* lung. A granuloma in the lung parenchyma (star) surrounded by areas of congestion and atelectasis (arrows) can be observed. Inset: AFB groups

within the granuloma (immersion) (black arrows). (Ziehl-Neelsen. 400x)


**Table 1.** Isolation of *Mycobacterium spp / M. pinnipedii* in *Arctocephalus australis*, *Otaria flavescens* and *Mirounga leonina* in Uruguay.

Macro and microscopic lesions were recorded. Hydrothorax and hemothorax were found in two adult animals. Papillary and proliferative lesions in parietal and visceral pleura (Fig. 7) were associated with a chronic inflammatory process, mononuclear and lymphocytic infiltration, and in some cases congestion and hemorrhage. Lungs presented yellowishwhite nodules on surface and deep pulmonary parenchyma, which corresponded to granulomas with AFB. Histology showed mononuclear infiltration throughout the parenchyma as well as congestion, emphysema and atelectasia near nodules. Granulomas showed the typical structure with a necrotic center surrounded by a mild fibroblastic reaction and mononuclear infiltration with AFB in single arrangements or small groups (Fig. 8). In bronchi and bronchioles mononuclear exudates at the lumen and lymphocyte/macrophage aggregates below the cartilage were present. Necrotic foci were also recorded in mediastinal lymph nodes with AFB. In one juvenile sea lion a mediastinal abscess and hematoma were found between the great vessels near the heart, which was associated with a chronic inflammatory process.

(Castro-Ramos et al., 2005, 2006) (Fig. 9).

sampling

*Mirounga leonina* in Uruguay.

associated with a chronic inflammatory process.

Code Year of

strains were isolated. Isolates were also obtained from organs without gross lesions belonging to pups and juvenile fur seals (n = 9) and from a sub-adult male elephant seal

9/2001 2001 Aa ♂ Adult w --- --- 2493 2002 Of ♀ Juvenile w + + 0874 2002 Aa ♂ Adult w + + 0873 2003 Aa ♀ Pup w + + 0875 2003 Aa ♀ Pup w + + 1405 2003 Of ♂ Juvenile w + + 1332/3 2004 Aa ♀ Pup w --- + 2172/1 2005 Aa ♀ Pup w - + 2172/2 2005 Aa ♀ Pup w - + 2172/3 2005 Aa ♀ Pup w - + 2172/4 2005 Aa ♂ Pup w - + 2172/6 2005 Aa ♂ Pup w - + 2172/7 2005 Aa ♂ Pup w - + 2173 2005 Of ♀ Pup w - + 2174 2005 Ml ♂ Juvenile w - +

Aa= *Arctocephalus australis*; Of= *Otaria flavescens*; Ml= *Mirounga leonina*; c= captive, w= wild.

Macro and microscopic lesions were recorded. Hydrothorax and hemothorax were found in two adult animals. Papillary and proliferative lesions in parietal and visceral pleura (Fig. 7) were associated with a chronic inflammatory process, mononuclear and lymphocytic infiltration, and in some cases congestion and hemorrhage. Lungs presented yellowishwhite nodules on surface and deep pulmonary parenchyma, which corresponded to granulomas with AFB. Histology showed mononuclear infiltration throughout the parenchyma as well as congestion, emphysema and atelectasia near nodules. Granulomas showed the typical structure with a necrotic center surrounded by a mild fibroblastic reaction and mononuclear infiltration with AFB in single arrangements or small groups (Fig. 8). In bronchi and bronchioles mononuclear exudates at the lumen and lymphocyte/macrophage aggregates below the cartilage were present. Necrotic foci were also recorded in mediastinal lymph nodes with AFB. In one juvenile sea lion a mediastinal abscess and hematoma were found between the great vessels near the heart, which was

**Table 1.** Isolation of *Mycobacterium spp / M. pinnipedii* in *Arctocephalus australis*, *Otaria flavescens* and

Species Sex Category Origin Baciloscopy Culture

**Figure 7.** Macroscopic view of an *Arctocephalus australis* lung. Numerous granulomas were present on the surface and deep parenchyma (white arrows) and a significant thickening of the visceral pleura.

**Figure 8.** Histological section from an *Arctocephalus australis* lung. A granuloma in the lung parenchyma (star) surrounded by areas of congestion and atelectasis (arrows) can be observed. Inset: AFB groups within the granuloma (immersion) (black arrows). (Ziehl-Neelsen. 400x)

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 167

skin test). Most individuals of the population carrying the dormant bacilli may become ill with TB at some point in their life, if an immunodeficient situation develops. It is therefore necessary to consider all infected individuals as potentially diseased animals (Rider, 1999). The fact that most gross and microscopic lesions are located in the respiratory system of pinnipeds (Bastida et al., 1999; Bernardelli et al., 1996; Castro-Ramos et al., 1998, 2001; Forshaw & Phelps, 1991; Katz et al., 2002; Moser et al., 2008; Woods et al., 1995) indicates that the main transmission is by aerosols. Less frequently, lesions were located in liver, spleen, kidney and abdominal lymph nodes (Bernardelli et. al., 1996; Forshaw & Phelps 1991; Kiers et al., 2008; Kriz et al., 2011), cases in which the bacilli probably spread through blood flow or swallowing of sputum containing mycobacteria (Forshaw & Phelps, 1991; Kriz et al., 2011). In wildlife, it is expected that transmission takes place mainly through spray during coughing and sneezing which is frequently observed in pups and adults (H. Katz,

*M. pinnipedii* have been isolated from fur seals, sea lions and elephant seals from the Southern Hemisphere (*Arctocephalus australis, A. forsteri, A. pussilus doriferus, A. tropicalis, Otaria flavescens, Neophoca cinerea, Mirounga leonina, Phocarctos hookeri*). They have been found both in wild animals and specimens kept in European aquaria or theme parks taken from South American colonies (Bernardelli et al., 1996; Castro-Ramos et al., 2005; Cousins et al., 2003; Duignan et al., 2003; Kiers et al., 2008; Kriz et al., 2011; Lacave, 2009). Micobacteriosis was diagnosed in only one Otariid species of the Northern Hemisphere (California sea lion, *Zalophus californianus*, Ehlers, 1965; Gutter et al., 1987). Most diagnoses were made in captive *Otaria flavescens*, probably because it is the most common species in aquaria as it is easily trained. Uruguay exported this species destined for aquaria from 1980 to 2006, when captures were restricted due to population decline (Páez, 2006). Nowadays, only live juvenile specimens of *A. australis* are caught for exportation to different destinations (Asia, Latin America and Europe). As this species is also carrier of *M. pinnipedii* (Cousins et al., 2003; Castro-Ramos et al., 2006; Katz et al., 2002) it is important to establish accurate diagnostic methods. The isolation of *M. pinnipedii* from different mammalian species (cattle, Bactrian camels, Malayan tapirs, Guinea pigs and humans), indistinguishable from strains isolated from pinnipeds, suggests that the bacillus has significant potential to infect a wide range of hosts, particularly when animals are in captivity (Cousins et al., 2003;

Presently, the diagnostic methods in living specimens have certain inaccuracies or deficiencies that make it difficult to establish a universal technique or golden standard. This is particularly important given that individuals carrying the microorganism can take years to show signs of the disease; only in terminal cases have nonspecific symptoms including anorexia, dysphagia, lethargy and weight loss been described. Coughing has not been described as a sign accompanying respiratory infection although significant lung lesions were present (Bernardelli, 1996; Castro-Ramos et al., 1998, 2006; Cousins et al., 1993; Kiers et al., 2008; Kriz et al., 2011; Lacave, 2009). In necropsied seals, tuberculosis diagnosis had been made based on mycobacterial isolation, histopathology and genetic characterization of strains. Imaging methods (radiography, computer tomography) have been used in captive

pers. obs.) or the typical naso-nasal contact between pinnipeds.

Kiers et al., 2008; Moser et al., 2008).

**Figure 9.** *Mycobacterium* spp. */ M. pinnipedii* colonies in Stonebrink medium culture. Sample obtained from a *Mirounga leonina* lung.

These results indicate a wide distribution of the disease in Uruguayan fur seal and sea lion colonies associated with a high prevalence of the disease in zoos and aquaria from South American native pinnipeds populations (Jurzinsky et al., 2011; Lacave, 2009; Lacave et al., 2009; Moser et al., 2008). The region used for genetic analysis can only discriminate between two large complexes within the Genera *Mycobacterium*, *Mycobacterium tuberculosis* complex (MTC) and *M. avium* (MAC). Based on these results we can conclude that the sequences studied are part of the MTC and correspond to *M. pinnipedii* as determined by more precise genetic studies (Cousins et al., 2003, Jurczinski et al., 2012; Kriz et al., 2011; Moser et al., 2008) which include a strain isolated in Uruguay (Cousins et al., 2003). Discrimination between different members of the MTC is essential for epidemiological investigations of wild populations, as well as the diagnosis of human cases associated with an adequate chemotherapy (de la Rua-Domenech, 2006).

The isolation of bacilli in pups and juvenile animals without apparent lesions indicates early transmission of the organism even though the animals do not show symptoms of respiratory disease. Similar findings were reported for wild carnivores for which positive cultures for *M. bovis* were not accompanied by gross or histological lung lesions (Bruning-Fann et al., 2001; Little et al., 1982). Furthermore, cattle with *M. bovis* developed a non-progressive disease, with small lesions in lymph nodes (retropharyngeal and mediastinal), which in turn are positive reactors to diagnostic tests for delayed cutaneous hypersensitivity (Tuberculin skin test). Most individuals of the population carrying the dormant bacilli may become ill with TB at some point in their life, if an immunodeficient situation develops. It is therefore necessary to consider all infected individuals as potentially diseased animals (Rider, 1999).

166 New Approaches to the Study of Marine Mammals

from a *Mirounga leonina* lung.

chemotherapy (de la Rua-Domenech, 2006).

**Figure 9.** *Mycobacterium* spp. */ M. pinnipedii* colonies in Stonebrink medium culture. Sample obtained

These results indicate a wide distribution of the disease in Uruguayan fur seal and sea lion colonies associated with a high prevalence of the disease in zoos and aquaria from South American native pinnipeds populations (Jurzinsky et al., 2011; Lacave, 2009; Lacave et al., 2009; Moser et al., 2008). The region used for genetic analysis can only discriminate between two large complexes within the Genera *Mycobacterium*, *Mycobacterium tuberculosis* complex (MTC) and *M. avium* (MAC). Based on these results we can conclude that the sequences studied are part of the MTC and correspond to *M. pinnipedii* as determined by more precise genetic studies (Cousins et al., 2003, Jurczinski et al., 2012; Kriz et al., 2011; Moser et al., 2008) which include a strain isolated in Uruguay (Cousins et al., 2003). Discrimination between different members of the MTC is essential for epidemiological investigations of wild populations, as well as the diagnosis of human cases associated with an adequate

The isolation of bacilli in pups and juvenile animals without apparent lesions indicates early transmission of the organism even though the animals do not show symptoms of respiratory disease. Similar findings were reported for wild carnivores for which positive cultures for *M. bovis* were not accompanied by gross or histological lung lesions (Bruning-Fann et al., 2001; Little et al., 1982). Furthermore, cattle with *M. bovis* developed a non-progressive disease, with small lesions in lymph nodes (retropharyngeal and mediastinal), which in turn are positive reactors to diagnostic tests for delayed cutaneous hypersensitivity (Tuberculin The fact that most gross and microscopic lesions are located in the respiratory system of pinnipeds (Bastida et al., 1999; Bernardelli et al., 1996; Castro-Ramos et al., 1998, 2001; Forshaw & Phelps, 1991; Katz et al., 2002; Moser et al., 2008; Woods et al., 1995) indicates that the main transmission is by aerosols. Less frequently, lesions were located in liver, spleen, kidney and abdominal lymph nodes (Bernardelli et. al., 1996; Forshaw & Phelps 1991; Kiers et al., 2008; Kriz et al., 2011), cases in which the bacilli probably spread through blood flow or swallowing of sputum containing mycobacteria (Forshaw & Phelps, 1991; Kriz et al., 2011). In wildlife, it is expected that transmission takes place mainly through spray during coughing and sneezing which is frequently observed in pups and adults (H. Katz, pers. obs.) or the typical naso-nasal contact between pinnipeds.

*M. pinnipedii* have been isolated from fur seals, sea lions and elephant seals from the Southern Hemisphere (*Arctocephalus australis, A. forsteri, A. pussilus doriferus, A. tropicalis, Otaria flavescens, Neophoca cinerea, Mirounga leonina, Phocarctos hookeri*). They have been found both in wild animals and specimens kept in European aquaria or theme parks taken from South American colonies (Bernardelli et al., 1996; Castro-Ramos et al., 2005; Cousins et al., 2003; Duignan et al., 2003; Kiers et al., 2008; Kriz et al., 2011; Lacave, 2009). Micobacteriosis was diagnosed in only one Otariid species of the Northern Hemisphere (California sea lion, *Zalophus californianus*, Ehlers, 1965; Gutter et al., 1987). Most diagnoses were made in captive *Otaria flavescens*, probably because it is the most common species in aquaria as it is easily trained. Uruguay exported this species destined for aquaria from 1980 to 2006, when captures were restricted due to population decline (Páez, 2006). Nowadays, only live juvenile specimens of *A. australis* are caught for exportation to different destinations (Asia, Latin America and Europe). As this species is also carrier of *M. pinnipedii* (Cousins et al., 2003; Castro-Ramos et al., 2006; Katz et al., 2002) it is important to establish accurate diagnostic methods. The isolation of *M. pinnipedii* from different mammalian species (cattle, Bactrian camels, Malayan tapirs, Guinea pigs and humans), indistinguishable from strains isolated from pinnipeds, suggests that the bacillus has significant potential to infect a wide range of hosts, particularly when animals are in captivity (Cousins et al., 2003; Kiers et al., 2008; Moser et al., 2008).

Presently, the diagnostic methods in living specimens have certain inaccuracies or deficiencies that make it difficult to establish a universal technique or golden standard. This is particularly important given that individuals carrying the microorganism can take years to show signs of the disease; only in terminal cases have nonspecific symptoms including anorexia, dysphagia, lethargy and weight loss been described. Coughing has not been described as a sign accompanying respiratory infection although significant lung lesions were present (Bernardelli, 1996; Castro-Ramos et al., 1998, 2006; Cousins et al., 1993; Kiers et al., 2008; Kriz et al., 2011; Lacave, 2009). In necropsied seals, tuberculosis diagnosis had been made based on mycobacterial isolation, histopathology and genetic characterization of strains. Imaging methods (radiography, computer tomography) have been used in captive animals, but in wild conditions these procedures are impractical. Chest radiographs were performed on pinnipeds of different sizes, but in cases of very small lesions in large animals with thick blubber, radiological images do not give appropriate information (Forshaw & Phelps, 1991; Jurczynski et al., 2012). In some zoos computer tomography has been used for detection of small calcified granulomas (Jurczynski et al., 2011, 2012), but it is very difficult to be used routinely. Different serological tests (rapid test, Elephant TB STAT-PAK, Chembio; multiantigen print immunoassay (MAPIA) Chembio; dual path platform assay (DPP Vet; Chembio) have been used in *O. flavescens* individuals in captivity, with DPP technique demonstrating greater sensitivity (87.5%) (Jurczynski et al., 2012). The tuberculin skin test (TST) with purified protein derivative (PPD bovine and avian) for screening has been done in *O. flavescens* and *A. australis* individuals (Bernardelli et al., 1990; Castro-Ramos et al., 1998; Kiers et al, 2008; Lacave, 2009) and reported by Forshaw & Phelps (1991) in *A. forsteri, A. pusillus doriferus* and *N. cinerea*. This technique is very sensitive, economical and easy to perform. The possible occurrence of false negatives must be taken into consideration in cases of advanced infection with anergy or very recent infections that have not yet generated an appropriate immune response (Jurczynski et al., 2011). False positives may also occur in nonspecific cases of exposure to non-tuberculous mycobacteria (*Mycobacterium avium, M. chelonae, M. fortuitum* and *M. smegmatis*) (Bernardelli et al., 1990; Forshaw & Phelps, 1991). Tissue and bronchial secretion smears have been used for diagnosis (Jurczynski et al., 2012), but the AFB may correspond to mycobacteria other than *M. pinnipedii* and, therefore, other confirmation methods must be used. Molecular techniques (PCR, spoligotyping and MIRU / VNTR) applied to samples from purified cultures, tissues and sputum, have produced quick results, allowing the identification of strains involved and their origin (Cousins et al., 2003; Jurczynski et al., 2011; Kiers et al., 2008; Kriz et al., 2011; Moser et al., 2008). In zoo collections, it is suggested that the final diagnosis should be based on the simultaneous use of three different methods, needing a minimum of two positive tests to increase the overall sensitivity when making the final decision for euthanasia. In case of wild animals, the possible diagnostic methods could include the TST, culture and molecular analysis of sputum (PCR) and serology (DPP). This would be extremely important in order to establish or confirm the endemic conditions of the disease in wild colonies and prevent the exportation or handling of carrier animals.

Uruguayan Pinnipeds (*Arctocephalus australis* and *Otaria flavescens*): Evidence of Influenza Virus and *Mycobacterium pinnipedii* Infections 169

fungal and parasitic agents, provoking diseases that result in mass mortality events (Gulland et al., 1996; Miller et al., 2004; Ostehaus, 2000). Once new or previous pathogens are established in the host, they can represent a health risk to other marine vertebrates, humans, or both. It is, however, very difficult to know which routes these zoonotic marine

The dynamic governing the relationship between infectious diseases affecting humans and marine vertebrates, including sea mammals is very complex and generally poorly understood. It is probable that human activity is a greater threat to marine vertebrate health than vice versa (Mos et al., 2006). However, it is very difficult to establish the role of marine animals as vectors or carriers of zoonotic diseases. Although the role of sea mammals in transmission of potential zoonotic pathogens is not well established, several risk factors, including frequent and prolonged direct contact with live specimens, were clearly identified in workers exposed to these animals (Hunt et al. 2008). Thus, these animals may present a zoonotic potential and also the potential for epizootic events which could cause health problems in marine animals. Since the influenza virus is transmitted by direct contact with infected individuals, by aerosol, or contact with infected objects, several incidents of influenza A virus transmission have occurred from infected seals and whales maintained in

Due to their life span, high trophic feeding and the continuous exposition to emerging pathogens, sea mammals might be considered as sentinel species of emerging/re-emerging diseases. Pinnipeds share these characteristics and may serve as effective sentinels providing information about public and aquatic ecosystem health, and indicating the current or potential negative impacts on animal health at the individual or population level. Implementing a proper management and the accurate execution of conservation policies of wildlife requires the analysis of the vulnerability of the animals to infectious diseases*.* The majority of animals included in our studies belonged to colonies near cities or towns in proximity to shore (Punta del Este, Polonio's Cape), regions that are commonly used by humans, marine and terrestrial mammals and different avian species, establishing an optimal opportunity for zoonotic disease transmission and long-term disease maintenance. The dense aggregations of pinniped colonies make fur seals and sea lions vulnerable to epizootics. Also, movements between adjacent latitudinal domains are common in both pinniped species, which could transmit or acquire pathogens during feeding trips to other

The diagnosis of influenza virus in high-density seal populations in Uruguay generated considerable concern about the potential impacts on South American fur seal and sea lion colonies, as well as potential health risks to humans and domestic animals. In the evolution and ecology of influenza viruses, interspecies transmission is an important factor; seabirds and marine mammals are conspicuous animals that integrate changes in the ecosystem and reflect the existing state of the environment (Aguirre & Tabor, 2004; Boersma, 2008; Moore, 2008; Thiele et al., 2004). Transmission of the influenza virus occurs between avian and several marine mammal species *(*Mandler et al.*,* 1990*)* at least for influenza A virus,

mammal infections take in the marine environment.

captivity to humans (Webster et al.*,* 1981*).*

parts of the Atlantic Ocean and the La Plata River.
